The Journal of Physical Chemistry B
ARTICLE
and Asp259 side chains. The Ser117 nucleophile attacks the
Michigan Medical School for providing the cDNA samples. We
also acknowledge the Center for Computational Sciences at the
University of Kentucky for supercomputing time on the IBM
X-series cluster with 340 nodes or 1360 processors.
ζ
electron-deficient C atom of (þ)-cocaine benzoyl ester, form-
η
ing a tetrahedral intermediate in which the carbonyl oxygen (O )
of (þ)-cocaine with developing negative charge is stabilized by
two tyrosine residues (Tyr44 and Tyr118) in the oxyanion hole.
γ
ζ
Then His287 transfers a proton (H ) to the ester oxygen (O ) of
the leaving ecgonine group, completing the acylation stage.
The QM/MM-optimized geometries indicate that the oxya-
nion hole stabilizes the negative charge of the (þ)-cocaine
’
REFERENCES
(1) Mendelson, J. H.; Mello, N. K. N. Engl. J. Med. 1996, 334 (15),
965–972.
η
carbonyl oxygen (O ) developing during the hydrolysis by
(2) Paula, S.; Tabet, M. R.; Farr, C. D.; Norman, A. B.; Ball, W. J.
providing two hydrogen bonds with Tyr44 and Tyr118. The
hydrogen bond with Tyr44 is particularly strong and is the
J. Med. Chem. 2003, 47 (1), 133–142.
(3) Singh, S. Chem. Rev. 2000, 100 (3), 925–1024.
η
primary factor stabilizing the carbonyl oxygen (O ) of (þ)-
(4) Substance Abuse and Mental Health Services Administration.
Drug Abuse Warning Network, 2004: National Estimates of Drug-Related
Emergency Department Visits; DAWN Series D-28; DHHS Publication
No. SMA 06-4143; Office of Applied Studies, Department of Health &
Human Services: Rockville, MD, 2006.
cocaine benzoyl ester.
The highest energy barrier calculated for the acylation of (þ)-
cocaine is ∼9.1 kcal/mol, associated with the first reaction step of
acylation. The calculated energy barrier of ∼9.1 kcal/mol is
much lower than the highest energy barrier for the deacylation
(5) Sparenborg, S.; Vocci, F.; Zukin, S. Drug Alcohol Depend. 1997,
48 (3), 149–51.
(
∼17.9 kcal/mol, associated with the first reaction step of
(
(
6) Gorelick, D. A. Drug Alcohol Depend. 1997, 48 (3), 159–65.
7) Gorelick, D. A.; Gardner, E. L.; Xi, Z. X. Drugs 2004, 64 (14),
deacylation). Therefore, the deacylation of (þ)-cocaine, which
is identical to that of (ꢀ)-cocaine, is rate-determining, revealing
that CocE-catalyzed hydrolyses of (þ)- and (ꢀ)-cocaine have a
common rate-determining step. All of these results predict that
1547–73.
(8) Dickerson, T. J.; Janda, K. D. Recent Advances for the Treatment
of Cocaine Abuse: Central Nervous System Immunopharmacotherapy.
In Drug Addiction; Rapaka, R. S., Sad ꢀe e, W., Eds; Springer: New York,
the catalytic rate constant (k ) of CocE against (þ)-cocaine
cat
2
008; pp 217ꢀ229.
9) Gao, D.; Cho, H.; Yang, W.; Pan, Y.; Yang, G.; Tai, H.-H.; Zhan,
C.-G. Angew. Chem., Int. Ed. 2006, 45 (4), 653–657.
10) Vocci, F. J.; Acri, J.; Elkashef, A. Am. J. Psychiatry 2005, 162 (8),
432–1440.
11) Zheng, F.; Yang, W.; Ko, M.-C.; Liu, J.; Cho, H.; Gao, D.; Tong,
should be the same as that of CocE against (ꢀ)-cocaine, in
contrast with the remarkable difference between human BChE-
catalyzed hydrolyses of (þ)- and (ꢀ)-cocaine. The computa-
tional prediction has been confirmed by performing experimen-
tal kinetic analysis on CocE-catalyzed hydrolysis of (þ)-cocaine,
for the first time, in comparison with CocE-catalyzed hydrolysis
of (ꢀ)-cocaine.
(
(
1
(
M.; Tai, H.-H.; Woods, J. H.; Zhan, C.-G. J. Am. Chem. Soc. 2008, 130
(36), 12148–12155.
(12) Pan, Y.; Gao, D.; Yang, W.; Cho, H.; Yang, G.; Tai, H.-H.; Zhan,
C.-G. Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (46), 16656–16661.
The determined common rate-determining reaction step and
detailed mechanistic differences in the acylation between CocE-
catalyzed hydrolyses of (þ)- and (ꢀ)-cocaine provide a valuable
mechanistic base for future rational design of CocE mutants with
an improved catalytic activity against cocaine. In particular, the
common rate-determining reaction step indicates that rational
design of a high-activity mutant of CocE should be focused on
stabilization of the transition-state structure (TS3) for the first
reaction step of the deacylation. The mutation-caused stabiliza-
tion of the transition state for the rate-determining reaction step
could lead to a decrease in the overall energy barrier and, thus, an
increase in the catalytic rate constant.
(
13) Bresler, M. M.; Rosser, S. J.; Basran, A.; Bruce, N. C. Appl.
Environ. Microbiol. 2000, 66 (3), 904–908.
14) Larsen, N. A.; Turner, J. M.; Stevens, J.; Rosser, S. J.; Basran, A.;
Lerner, R. A.; Bruce, N. C.; Wilson, I. A. Nat. Struct. Mol. Biol. 2002, 9
1), 17–21.
15) Cooper, Z. D.; Narasimhan, D.; Sunahara, R. K.; Mierzejewski,
(
(
(
P.; Jutkiewicz, E. M.; Larsen, N. A.; Wilson, I. A.; Landry, D. W.; Woods,
J. H. Mol. Pharmacol. 2006, 70 (6), 1885–1891.
(16) Ko, M.-C.; Bowen, L. D.; Narasimhan, D.; Berlin, A. A.; Lukacs,
N. W.; Sunahara, R. K.; Cooper, Z. D.; Woods, J. H. J. Pharmacol. Exp.
Ther. 2007, 320 (2), 926–933.
(
17) Emily, M. J.; Michelle, G. B.; Ziva, D. C.; Diwahar, N.; Roger,
K. S.; James, H. W. Ann. Emerg. Med. 2008, 54, 409–420.
18) Gao, D.; Narasimhan, D. L.; Macdonald, J.; Brim, R.; Ko, M.-C.;
’
AUTHOR INFORMATION
(
Corresponding Author
Landry, D. W.; Woods, J. H.; Sunahara, R. K.; Zhan, C.-G. Mol.
Pharmacol. 2009, 75 (2), 318–323.
*
Phone: (859) 323-3943. Fax: (859) 323-3575. E-mail: zhan@
(19) Brim, R. L.; Nance, M. R.; Youngstrom, D. W.; Narasimhan, D.;
uky.edu.
Zhan, C.-G.; Tesmer, J. J. G.; Sunahara, R. K.; Woods, J. H. Mol.
Pharmacol. 2010, 77 (4), 593–600.
Author Contributions
These authors contributed equally to this work.
†
(
20) Collins, G. T.; Brim, R. L.; Narasimhan, D.; Ko, M. C.;
Sunahara, R. K.; Zhan, C.-G.; Woods, J. H. J. Pharmacol. Exp. Ther.
009, 331 (2), 445–455.
21) Narasimhan, D.; Nance, M. R.; Gao, D.; Ko, M.-C.; Macdonald,
2
(
’
ACKNOWLEDGMENT
J.; Tamburi, P.; Yoon, D.; Landry, D. M.; Woods, J. H.; Zhan, C.-G.;
Tesmer, J. J. G.; Sunahara, R. K. Protein Eng. Des. Sel. 2010, 23, 537–547.
This work was supported in part by the National Institutes of
(22) Zhan, C.-G.; Gao, D. Q. Biophys. J. 2005, 89 (6), 3863–3872.
Health (Grants R01 DA025100, R01 DA021416, and R01
DA013930). The entire work was performed at the University
of Kentucky. X.Z. worked in C.-G.Z.’s laboratory for this project
at the University of Kentucky as an exchange graduate student
from Central China Normal University. We thank Drs. Roger K.
Sunahara and Diwahar L. Narasimhan at the University of
(23) Zhan, C.-G.; Zheng, F.; Landry, D. W. J. Am. Chem. Soc. 2003,
125 (9), 2462–74.
(24) Sun, H.; Pang, Y.-P.; Lockridge, O.; Brimijoin, S. Mol. Pharma-
col. 2002, 62 (2), 220–224.
(25) Gao, Y.; Atanasova, E.; Sui, N.; Pancook, J. D.; Watkins, J. D.;
Brimijoin, S. Mol. Pharmacol. 2005, 67 (1), 204–211.
5
024
dx.doi.org/10.1021/jp200975v |J. Phys. Chem. B 2011, 115, 5017–5025